Phylogeny and Evolution of Pharmacophagy in Tiger Moths (Lepidoptera: Erebidae: Arctiinae) Jennifer M. Zaspel1*, Susan J. Weller2,5, Charles T. Wardwell2, Reza Zahiri3, Niklas Wahlberg4 1 Department of Entomology, Purdue University, West Lafayette, Indiana, United States of America, 2 Department of Entomology, University of Minnesota, Saint Paul, Minnesota, United States of America, 3 Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada, 4 Laboratory of Genetics, Department of Biology, University of Turku, Turku, Finland, 5 Bell Museum of Natural History, University of Minnesota, Minneapolis, Minnesota, United States of America Abstract The focus of this study was to reconstruct a phylogenetic hypothesis for the moth subfamily Arctiinae (tiger moths, woolly bears) to investigate the evolution of larval and adult pharmacophagy of pyrrolizidine alkaloids (PAs) and the pathway to PA chemical specialization in Arctiinae. Pharmacophagy, collection of chemicals for non-nutritive purposes, is well documented in many species, including the model species Utetheisa ornatrix L. A total of 86 exemplar ingroup species representing tiger moth tribes and subtribes (68 genera) and nine outgroup species were selected. Ingroup species included the most species- rich generic groups to represent the diversity of host-plant associations and pharmacophagous behaviors found throughout Arctiinae. Up to nine genetic markers were sequenced: one mitochondrial (COI barcode region), one nuclear rRNA (D2 region, 28S rRNA), and seven nuclear protein-coding gene fragments: elongation factor 1-a protein, wingless, ribosomal protein subunit S5, carbamoylphosphate synthase domain regions, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and cytosolic malate dehydrogenase. A total of 6984 bp was obtained for most species. These data were analyzed using model-based phylogenetic methods: maximum likelihood (ML) and Bayesian inference (BI). Ancestral pharmacophagous behaviors and obligate PA associations were reconstructed using the resulting Bayes topology and Reconstructing Ancestral States in Phylogenies (RASP) software. Our results corroborate earlier studies on the evolution of adult pharmacophagous behaviors, suggesting that this behavior arose multiple times and is concentrated in the phaegopterine-euchromiine-ctenuchine clade (PEC). Our results suggest that PA specialization may have arisen early in the phylogeny of the subfamily and that facultative larval pharmacophagous behaviors are the derived condition. Citation: Zaspel JM, Weller SJ, Wardwell CT, Zahiri R, Wahlberg N (2014) Phylogeny and Evolution of Pharmacophagy in Tiger Moths (Lepidoptera: Erebidae: Arctiinae). PLoS ONE 9(7): e101975. doi:10.1371/journal.pone.0101975 Editor: Carlos Lo´pez-Vaamonde, Institut National de la Recherche Agronomique (INRA), France Received November 5, 2013; Accepted June 12, 2014; Published July 18, 2014 Copyright: ß 2014 Zaspel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for the study was provided by NSF-DEB 0919185 to S. Weller and J. Zaspel. Additional funds were provided by UMN Agricultural Experiment Station and Purdue University. The following Finnish foundations also provided support for this research: CIMO, Finnish Cultural Foundation and the Alfred Kordelin Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction Within the mostly phytophagous tribes Arctiini and Amerilini, larvae supplement endogenous defenses by sequestering com- Arctiinae (tiger moths and woolly bears) are a charismatic moth pounds from their host plants like pyrrolizidine alkaloids (PAs), lineage with a complex evolutionary relationship with plant and cardiac glycosides (CGs) or iridoid glycosides (IRs) [4], [8–10]. fungal chemistries. Comprised of approximately 11,000 species These noxious compounds are of great importance because they [1], this cosmopolitan group is well known among ecologists and have been implicated in severe human and animal diseases [2]. evolutionary biologists for the evolution of bright coloration and Additionally, through behavioral assays of vertebrate and inver- spectacular adult mimicry of wasps, beetles and unpalatable moths tebrate predators, these three classes of compounds (PAs, CGs, and butterflies (Figure 1). Both aposematic adults (Figure 1A) and and IRs) have been shown to deter predation [11], and of these larvae (Figure 1B–D) typically harbor endogenous biogenic classes of compounds, the acquisition, sequestration and dissem- amines, like histamines, which are often supplemented with ination of PAs is best understood [12]. secondary compounds acquired from larval hosts or through Larvae of PA specialist species (e.g., Utetheisa ornatrix, Tyria pharmacophagy - feeding on plants to obtain chemicals rather than jacobaeae (L.) [13], Creatonotos gangis (L.)) feed only on PA- nutrients [2]. Some larvae become pharmacophagous when containing plants and prefer foliage and seed pods that have the parasitized, and this ‘‘self-medication’’ improves survivorship [3]. highest concentration of these compounds [12], [14]. Larval PAs Adult pharmacophagy is linked to acquiring chemical constituents are stored in an inactive form for transfer to the adult stage. In of courtship pheromone and nuptial gifts that improve male some adult males, PAs are transformed into danaidol (and mating success [2], [4–7]. Adult pharmocophagy has been chemical analogs) for dispersal in courtship pheromones [7]. documented in other Lepidoptera, notably Danainae (Nympha- Within the Old World genus Creatonotos, PAs are morphogenic; lidae) [2]; however, larval pharmacophagy has only been the size and complexity of the male-pheromone dispersing documented in Arctiinae. structures (coremata) are dependent upon the PA concentrations consumed by larvae [15]. In the rattlebox moth, Utetheisa PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e101975 Evolution of Pharmacophagy in Tiger Moths Figure 1. Tiger moth adults/larvae, diversity. A. Diversity of adult habitus (photos courtesy: Rebecca Simmons); B. Woolly Bear, Pyrrharctia isabella (photo courtesy of Bill Conner); C. Milkweed tussock, Euchaetes egle (photo courtesy of Rebecca Simmons), D. Rattle box moth larva, Utetheisa ornatrix (photo courtesy Nancy Jacobson). doi:10.1371/journal.pone.0101975.g001 ornatrix, male mating success depends upon the presence and titer species that exhibit adult pharmacophagy occur in the Neotropics, of PAs in his pheromones [16–19]. In this species, the males with the exception of the Old World genus Amerila. transfer PAs to the female in the spermatophore [20], and the Despite numerous ecological and behavioral studies, our females gain predator protection for themselves and their eggs understanding of arctiine phylogeny and how these pharmaco- [21]. In the rattlebox moth, PAs are considered an ‘‘honest’’ good- phagous feeding behaviors evolved was limited [10]. A culmina- genes signal of the male’s ability to acquire and transport PAs [18], tion of a series of molecular and morphological studies led to the [22–23]. recent placement of the family Arctiidae as a subfamily Arctiinae In contrast to this PA-obligate system, where PA acquisition and of Erebidae [31–32] and all taxonomic ranks of older literature is use is linked across the larval and adult stages, several have been translated to this most recent taxonomy (Table 1) [33]. The only chemically documented, and many species have host records of comprehensive phylogenetic reconstruction for Arctiinae was PA-containing plants. In oligophagous or polyphagous species, based on adult and immature morphology from 40 exemplar larvae accept PA-containing plants as part of a broader diet. They species [34]. The results supported the monophyly of the do not transfer them to the adult stage or actively collect them as subfamily and three lineages, tribe Arctiini and the traditional adults. In some cases, the purpose of PA acquisition appears to be arrangement of a sister group relationship of Lithosiini and medicinal: larvae increase their PA diet when parasitized by Syntomini (Figure 3A). Their tree disagreed with Bendib and endoparasitoids [24–25]. Thus, parasitized larvae that self- medicate with PAs tend to have greater resistance to parasitoids and thus higher survivorship than those that do not [26]. The precise mechanism leading to higher survivorship is not under- stood. Higher titers of PAs have also been shown to delay larval development and incur other fitness tradeoffs [26–27]. This behavioral plasticity of facultative pharmacophagy is thought to permit polyphagous larvae to respond to a heterogeneous selective environment [3], [25]. Although larvae facultatively acquire PAs, they do not store them through metamorphosis and transmit these to the pupal and adult stages. Thus, in these cases, the PA pharmacophagy is restricted to the larval stage. In contrast, there are several species whose pharmacophagy of PAs only occurs during the adult stage (Figure 2). Many species (typically males) seek out PA-plants to feed at wounds or acquire PAs by regurgitating saliva, dissolving PA